The following description of the background of the invention is provided to aid in understanding the invention, but is not admitted to be or describe prior art to the invention.
Existing methods for determining ratios of biological molecules involve multiple steps and often require a large amount of time to perform. These methods often utilize two or more components, usually antibodies, specific for each of the biological molecules. Thus, two or more discrete assays need to be conducted to determine the ratio. Hence, these systems prolong the time required to determine the ratio and also accumulate reagent costs.
In addition, many of the existing methods for determining the concentrations of biological molecules utilize several components, usually antibodies or labeled antigens, at concentrations in excess of the concentration of the biological molecules in a sample. Non-competitive or sandwich assays function by the use of antibodies in excess of the biological molecules. Competitive immunoassays function through a competition of binding of a biological molecule and a labeled biological molecule for a limited concentration of antibody. Because some biological molecules, such as hemoglobin or cell receptors, occur at high concentrations in biological fluids, existing methods that require components to be in excess of the biological molecules are of limited application. In addition, samples generally require a dilution prior to assay.
Determining the ratio of biological molecules has proved to be an important indicator for many medical conditions and procedures. In particular, the determination of the ratio of related biological molecules is useful. Related biological molecules are formed in an organism when a biological molecule becomes modified. Biological molecules can become modified, for example, by covalent chemical alteration or by the reversible binding of molecules.
Biological molecules can become chemically modified in an organism in an intermolecular fashion. For example, hemoglobin, a blood-borne oxygen carrier in organisms, can become modified by glucose moieties when the blood stream contains high levels of glucose. In the blood stream, the aldehyde group of glucose condenses with valine of hemoglobin to form a Schiff base. This reversible reaction is followed by a virtually irreversible rearrangement in which the double bond shifts to C-2 of the sugar to give a stable fructose derivative of hemoglobin. Stryer, Biochemistry, 3rd Ed., W. H. Freeman and Co., New York 1988. Hemoglobin that is modified in this manner is referred to as hemoglobin A1-C.
In addition, biological molecules can be modified in an intramolecular fashion. For example, troponin I, which normally exists in a reduced form in muscle cells, is oxidized when it is released into the blood stream of organisms suffering from a myocardial infarction. In particular, cysteine moieties within a discrete troponin I molecule can oxidize to form an intramolecular disulfide linkage. Methods of detecting related forms of troponin I that are released from muscle cells after a myocardial infarction are disclosed in PCT publication WO 96/33415.
Biological molecules can also become reversibly modified when high-affinity ligands bind to them. Cell receptors, for example, which are presented on the surface of a cell, can bind natural ligands or synthetic ligands with equilibrium dissociation constants in the micromolar to picomolar range.
The invention relates in part to novel methods of rapidly determining the ratio of biological molecules. The invention also relates in part to a kit for determining the ratio of related biological molecules.
The invention increases the rate for determining ratios of biological molecules as compared to the rates of determining these ratios using existing methods. The invention increases the rate for determining ratios of biological molecules by reducing the number of steps required for measuring the ratio.
Applicant has discovered that the ratio of biological molecules can be rapidly detected without measuring the absolute concentrations of the biological molecules by using a binding molecule, preferably an antibody, that recognizes each of the biological molecules but binds only one of the biological molecules at a time.
FIG. 1, which depicts one embodiment of the invention, serves as an illustrative example for the rapid determination of the ratio of biological molecules. The number of steps are reduced by probing a sample with a first component that binds a fraction of each of the biological molecules of interest. When the concentration of the first component is less than the concentrations of the biological molecules, the first component binds the biological molecules in a ratio related to the ratio at which the biological molecules exist in solution.
In one embodiment, the binding of one of the biological molecules to the first component excludes the binding of the other, even though the first component has the capacity of binding each of the molecules independently. The distribution of the biological molecules bound to the first compound is a statistical distribution that is directly related to the distribution of biological molecules in the sample.
These two features of the first component, the multiple binding feature and the exclusive binding feature, allow the first component to bind the biological molecules in a ratio related to the ratio of the biological molecules in the sample. For example, if the first component can bind each of molecules A and B, and A and B exist in the sample at a 3 to 1 ratio, the bound first component will have bound A and B in a 3 to 1 ratio or nearly this ratio.
Biological molecules A and B bind to the first component in a ratio related to their ratio in the sample, the relative on rates of the A and B binding to the first component determining the final ratio of A and B bound to the first component. Thus, the ratio of A to B can be bound to the first component in a ratio that is proportional to the ratio of A to B existing in a sample.
The second component of the invention detects the complex formed between the first component and one of the biological molecules. This complex may be detected when the second component binds to only one of the biological molecules, e.g., A or B, or if the second component binds to the complex formed between one of the biological molecules and the first component. The latter instance may provide an advantage if the biological molecules exist at high concentrations in the sample with respect to the concentration of the second component, since the second component will bind the complex comprising one biological molecule and the first component and not the unbound biological molecule.
Once the second component binds the complex comprising the first component and a biological molecule, a signal can be measured from a reporter molecule linked to one of the components of the invention. This signal can be applied to a standard curve that relates the signal to a ratio of the biological molecules. The standard curve can be prepared by measuring the signal, by the methods described herein, for samples prepared with known ratios of the biological molecules.
When biological molecules do not bind to the first component with equal affinity, standard curves relating the ratio to a signal generated by one of the components, preferably the first component, can be utilized to determine the ratio of A to B in the sample. In addition, normalization factors can be utilized to determine the ratio of A to B in a sample.
The ratio of the biological molecules is determined most rapidly when the components and the sample are mixed together at the same time and in the same vessel. This approach minimizes the number of steps required to determine the ratio of biological molecules, and thereby represents an advantage over existing techniques for determining the ratio of biological molecules. In particular, applications of the methods and kits described herein relate in part to increasing the efficiency of monitoring drug delivery, monitoring the historic blood-glucose level in diabetic patients, and monitoring the time of myocardial infarction.
The rapid rate of determining the ratio of biological molecules can enhance the recovery of patients suffering from particular medical conditions. Proper treatment can be expedited since the diagnosis results can be determined in a rapid manner. In the case of heart attacks, for example, a rapid determination of the oxidized to reduced troponin I ratio will hasten the determination of the time of a myocardial infarction, and thereby expedite the administration of a proper treatment to the patient. Expediting the treatment of a patient will improve that patient""s recovery from the myocardial infarction.
The rapid rate of determining the ratio of related biological molecules can also enhance the delivery of a therapeutic drug to a patient. In the case of a drug that binds and blocks a cell surface receptor, a rapid determination of the free receptor to occupied receptor ratio can determine whether a larger or smaller dose of the drug should be delivered to the patient for an effective therapy.
Furthermore, the invention allows for the determination of ratios of related biological molecules that exist at high concentrations in a sample. Hemoglobin, for example, exists at high concentrations in a patient""s blood stream. Hemoglobin becomes hemoglobin A1-C when it is modified with glucose in the patient""s blood stream. One component of the invention can isolate a fraction of the total hemoglobin molecules (hemoglobin and hemoglobin A1-C) and a second component can isolate one of the related molecules (such as hemoglobin A1-C) to determine the ratio of these related molecules even when they exist at high concentrations in a sample. This application of the methods described herein is useful for diabetic patients since hemoglobin A1-C represents the average blood glucose concentration over periods of time longer than one day. Because diabetic patients often cannot accurately determine their blood glucose levels due to variable readings using the techniques currently available to them, the methods and kits of the invention provide for the accurate and rapid determination of the average blood glucose level for diabetic patients.
Thus in a first aspect, the invention features a method of determining a solution ratio of biological molecules. The method comprises the steps of: (a) contacting the biological molecules with (i) a first component having specific binding affinity to each biological molecule, where the biological molecules bind to the first component in a binding ratio related to the solution ratio of the biological molecules; (ii) contacting the biological molecules with a second component having specific binding affinity for one of the biological molecules; and (b) determining the amount of a complex comprising the biological molecule, the first component, and the second component present as a measure of the solution ratio.
The term xe2x80x9cbiological moleculesxe2x80x9d as used herein refers to two or more molecules that exist naturally or unnaturally in a biological organism or fluid or an environmental sample. The ratio is preferably measured for four or more biological molecules, more preferably measured for three biological molecules, and most preferably measured for two biological molecules. The biological molecules can be related or unrelated. Biological molecules can be related by virtue of modification of one of the biological molecules. Thus, related molecules can exist as an unmodified molecule and a modified molecule.
A biological molecule may be modified in at least two manners: (i) modified covalently in an intermolecular or intramolecular fashion, or (ii) modified reversibly with a high affinity molecule. The molecule may be modified covalently by the addition of another chemical moiety (i.e., hemoglobin modified by a glucose moiety). An example of a molecule modified by a reversibly binding affinity molecule is a free receptor bound by an affinity ligand. The ligand may be a naturally occurring binding molecule of the free receptor or may alternatively be a synthetic ligand.
When the biological molecules of interest do not exist in a sample, the methods and kits of the invention can determine that the biological molecules do not exist in the sample. These types of results often yield useful information. For example, a determination that a blood sample contains negligible amounts of oxidized troponin I, might indicate that the patient from which the blood sample was taken has not suffered myocardial infarction. This type of result could save a hospital and patient from making large expenditures on health care for a condition which never existed. Thus, even when the methods of the present invention yield a negative result, the results monitored by the methods and kits of the invention are useful.
Examples of biological molecules include, but are not limited to organic and inorganic molecules, drugs, peptides, nucleic acids, receptors, cells and proteins.
The term xe2x80x9creceptorxe2x80x9d as used herein refers to a nonprotein or a protein component that binds specifically or nonspecifically to a molecule. Examples of receptors include, but are not limited to cell surface receptors, antibodies, binding proteins, binding fragments, avidin, non-protein templates and biomimetic receptors.
The term xe2x80x9ccomponentxe2x80x9d as used herein refers to a molecule that specifically binds to one or more of the biological molecules. The component preferably comprises a protein or polypeptide or peptide, more preferably comprises a peptidomimetic or organic compound, and most preferably comprises an antibody.
The term peptidomimetic as used herein refers to a peptide-like molecule containing non-hydrolyzable chemical moieties in place of one or more hydrolyzable moieties existing in naturally occurring peptides. Thus, regions of a peptide which are hydrolyzable, such as carboxyl moieties, are replaced by non-hydrolyzable moieties, such as methylene moieties, in a peptidomimetic.
The term xe2x80x9cantibodyxe2x80x9d as used herein refers to a monoclonal antibody, a polyclonal antibody, a binding fragment of an antibody, and a recombinant antibody. The term xe2x80x9cantibodyxe2x80x9d also refers to a receptor protein that can specifically bind to a target.
The terms xe2x80x9cspecific binding affinityxe2x80x9d or xe2x80x9cspecifically bindsxe2x80x9d or xe2x80x9cspecifically boundxe2x80x9d as used herein describe a component of the invention, preferably comprising, consisting of, or consisting essentially of an antibody, that binds to one or more biological molecules with greater affinity than it binds to other molecules under specified conditions. For instance, a first component of the invention may comprise a binding moiety having specific binding affinity for hemoglobin and hemoglobin A1-C; the binding moiety will not appreciably bind to molecules that are not hemoglobin or hemoglobin A1-C. Preferably, a component of the invention binds to a molecule with a specific binding affinity at least 5 times greater than it binds to other molecules, more preferably 10 times or 50 times greater than it binds to other molecules, and most preferably 100 times greater than it binds to other molecules.
The term xe2x80x9cbinding moietyxe2x80x9d as used herein refers to a molecule that comprises a component of the invention having specific binding affinity for a biological molecule or biological molecule/first component complex. The binding moiety is preferably a protein, polypeptide, or peptide, more preferably a peptidomimetic or organic compound, and most preferably an antibody.
Methods of binding and determining the amount of antibodies bound to a target in a sample are well-known to those skilled in the art. Harlo and Lane, Antibodies, A Laboratory Manual, 1989, Cold Spring Harbor Laboratories. The components that bind to the biological molecules of the invention can be monitored using techniques known to those skilled in the art. These techniques include manual applications and applications involving mechanical and electronic instrumentation.
The first component of the invention can bind to the biological molecules in a binding ratio related to the solution ratio of the biological molecules. The term xe2x80x9crelatedxe2x80x9d refers to solution ratios and binding ratios that are equal or nearly equal to one another. The solution ratio and binding ratio are nearly equal to one another when the ratio of the solution ratio to the binding ratio is between 0.1 and 10, preferably between 0.2 and 5, more preferably between 0.5 and 2, and most preferably equal to 1.
The solution ratio is also related to the binding ratio of the biological molecules when (i) one of the biological molecules can bind to the first component at one time, and (ii) each biological molecule has a similar on rate and a similar equilibrium constant for binding the first component. Thus, the binding of one of the biological molecules excludes the binding of another biological molecule also having specific binding affinity to the first component. This feature of the invention allows the first component to bind the biological molecules of interest in the same or related ratio as the molecules exist in the sample being probed with the components of the invention. These conditions allow the binding ratio to be of the same or related value to the solution ratio.
The term xe2x80x9ccomplexxe2x80x9d as used herein refers to two or more discrete molecules bound to one another in a non-covalent manner. Thus, a complex, for example, can comprise a biological molecule bound to a first component of the invention. The complex may also consist of or consist essentially of the first component bound to one biological molecule. In addition, a complex may exist that comprises, consists of, or consists essentially of a biological molecule and a first component. Furthermore, a complex may exist that comprises, consists of, or consists essentially of a biological molecule, a first component, and a second component. If the first component comprises an antibody, the first component may form a complex comprising, consisting of, or consisting essentially of the first component and two distinct types of biological molecules, due to the dual binding capacity of antibodies. Similarly, if the first component is an antibody, the first component may form a complex comprising, consisting of, or consisting essentially of the first component, two distinct types of biological molecules, and a second component. An antibody may also bind two molecules of the same type of biological molecule. Thus the complexes may contain two molecules of the same type of biological molecule.
A complex may be stable with respect to dilution of the free molecules comprising the complex when the molecules comprising the complex bind to one another with high affinity. High affinity interactions between the molecules of the complex can be achieved by noncovalent interactions, for example, such as electrostatic interactions, hydrophobic interactions, Van der Waals interactions, and hydrogen bond interactions.
The term xe2x80x9camountxe2x80x9d as used herein refers to an indication of the presence of a complex comprising, consisting of, or consisting essentially of a biological molecule, a first component, and a second component. The amount may be expressed, for example, in terms of an absorbance change or a change in fluorescent emission measured at one or more wavelengths in the ultraviolet, visible, or infrared range of wavelengths. An optical density or a fluorescent reading may be calculated into a ratio using a standard curve of the invention, as described herein by example. The amount can be assessed directly from a signal generated from one of the components themselves or by a separate component that specifically binds to the complex, which comprises, consists of, or consists essentially of a biological molecule, a first component, and a second component.
The term xe2x80x9cratioxe2x80x9d as used herein refers to the fraction of biological molecules. The ratio, for example, may represent the fraction of modified molecule to unmodified molecule. The ratio of these biological molecules may be expressed by the following fractions:
[unmodified molecule]/[modified molecule];
[modified molecule]/[unmodified molecule];
[unmodified molecule]/[modified molecule+unmodified molecule]; and
[modified molecule]/[modified molecule+unmodified molecule].
The ratio may also be determined for multiple biological molecules. For example a ratio might be determined for one biological molecule A to three other biological molecules B C, and D. This ratio could be determined by using a first component that binds to each of A, B, C, and D, where the binding of any one of A, B, C, or D excludes the binding of any of the others. The second component would have specific binding affinity for A. The ratio could be expressed as:
[A]/[B+C+D] or [A]/[A+B+C+D].
Likewise, the ratio of biological molecule B to the biological molecules A, C, and D can be measured using a second component having specific binding affinity for B. The ratio for this relation can be expressed as:
[B]/[A+C+D] or [B]/[A+B+C+D]
In general, the ratio of two or more biological molecules can be determined using the novel teachings described herein.
The term xe2x80x9csolution ratioxe2x80x9d as used herein refers to the ratio of the biological molecules as they exist in solution. The solution ratio may be the same ratio or a different ratio than the ratio of the biological molecules bound to the first component.
The term xe2x80x9cbinding ratioxe2x80x9d as used herein refers to the ratio of the biological molecules bound to the first component of the invention. The biological molecules may bind to the first component in the same or different ratios as they exist in the sample. Therefore, the binding ratio of the biological molecules may be the same or different than the solution ratio of the biological molecules.
The solution ratio is similar to the binding ratio of the biological molecules where (i) one of the biological molecules can bind to the first component at one time, and (ii) the biological molecules bind to the first component with a similar on rate and a similar equilibrium constant for binding the first component. Thus, the binding of one of the biological molecules excludes the binding of another biological molecule also having specific binding affinity to the first component.
The term xe2x80x9csimilar equilibrium constantxe2x80x9d refers to equilibrium dissociation constants for the first molecule binding to each of the biological molecules within a five-fold difference with respect to one another. This feature of the invention allows the first component to bind the biological molecules of interest in the same or different ratios as the molecules exist in the sample being probed with the components of the invention. These conditions allow the binding ratio to be of the same or similar value to the solution ratio.
In a preferred embodiment, one or more components 30 may have specific binding affinity for an epitope that consists of, or consists essentially of, a portion of a biological molecule and a portion of another component. For example, in FIG. 1A, the second component may have specific binding affinity for a portion of molecule B and a portion of component 1. This example also applies to FIGS. 1B and 1C. Examples of components, such as antibodies, that have specific binding affinity for an epitope that consists of a binding interface for two other molecules are well known in the art.
The term xe2x80x9cepitopexe2x80x9d as used herein can refer to a surface to which a component of the invention has specific binding affinity. An epitope can be a portion of a molecule of any size. An epitope can also be a portion of one molecule and a portion of one other molecule, where the two molecules bind to one another in a complex. An epitope on such a complex can consist of a region of one molecule and a region of another molecule that are adjacent and are located at a binding interface of the two molecules.
In another preferred embodiment the invention relates to the method of determining the ratio of biological molecules where the method further comprises one or more other distinct first components having specific binding affinity to other distinct biological molecules. By utilizing multiple distinct first components, the invention provides for a method of determining two or more ratios of biological molecules. For example, one first component can be utilized to measure the ratio of biological molecules A and B in a sample, and in the same sample, a different first component can determine the ratio of biological molecules C and D. This example can be readily modified by a person of ordinary skill in the art to include measuring multiple ratios of biological molecules using multiple first components of the invention.
In another preferred embodiment the invention relates to the method of determining the ratio of biological molecules, where the method further comprises one or more other distinct second components each having specific binding affinity for distinct biological molecules. Each distinct second component has specific binding affinity for only one biological molecule. Multiple second components can be utilized in conjunction with either one first component or multiple first components. An example of the former application is provided herein by example with respect to measuring ratios of biological molecules important for thrombosis. An illustration of using multiple second components in conjunction with one first component is presented in FIG. 1D. A protocol that entails the utilization of one first component in conjunction with two or three second components is provided herein by example. An illustration of utilizing multiple second components in conjunction with multiple first components is presented in FIG. 1C.
When the ratio of three or more biological molecules is measured using only one first component, the solution ratio and binding ratios are nearly equal to one another when the ratio of the solution ratio to the binding ratio is between 0.1 and 10, preferably between 0.2 and 5, more preferably between 0.5 and 2, and most preferably equal to 1.
In a preferred embodiment, the invention relates to the method of determining the ratio of biological molecules, where at least one of the components comprises an antibody. In this preferred embodiment, the first component can comprise an antibody, the second component can comprise an antibody, or the first component and the second component can comprise antibodies. Thus, the method can utilize a first component that comprises an antibody and a second component that comprises another type of polypeptide or organic molecule. Alternatively, the method may relate to two components that comprise antibodies.
In yet another preferred embodiment, the invention relates to the method of determining the ratio of biological molecules, where at least one of the components comprises a reporter molecule. In this preferred embodiment, the first component can comprise a reporter molecule, the second component can comprise a reporter molecule, or the first component and the second component can comprise reporter molecules. If both components comprise reporter molecules, the reporter molecules may be the same types of molecules, or preferably, different types of reporter molecules.
The term xe2x80x9creporter moleculexe2x80x9d as used herein refers to a signal generator or a signal generating element. These terms can refer to a number of elements: enzymes and their resultant effects on a substrate, colloidal metal particles, latex and silica particles with dye incorporated, and dye particles are examples of signal generators. An enzyme can catalyze the turnover of a substrate to produce a product that is detectable, for example, by absorbance or fluorescence technologies (e.g., ultra-violet, visible, infrared) or detectable by shift in pH. Reporter molecules may be linked to components of the invention, in particular antibodies, by techniques well-known to those skilled in the art. See e.g., Harlo and Lane, Antibodies, a Laboratory Manual, 198, Cold Spring Harbor Laboratories for examples of methods used to link reporter molecules to antibodies and other proteins as well as examples of various reporter molecules commonly used by those skilled in the art. The linkage can be a chemical moiety of varying length. The components of the invention may be modified with a reporter molecule either before the components are added to a sample comprising the biological molecules under study, or alternatively, after the components are added to the sample being probed with the components of the invention.
In another preferred embodiment the invention relates to the method of determining the ratio of biological molecules, where at least one component comprises a specific recognition moiety. In this preferred embodiment, the first component can comprise a specific recognition moiety, the second component can comprise a specific recognition moiety, or the first component and the second component can comprise specific recognition moieties. If both components comprise specific recognition moieties, the specific recognition moieties may be different from one another or the same recognition moiety.
The term xe2x80x9cspecific recognition moietyxe2x80x9d as used herein refers to a molecule covalently linked to a component of the invention which can be recognized by another binding molecule. The specific recognition moiety can be a peptide, polypeptide, protein, or a non-peptide molecule. An example of such a specific recognition moiety is a peptide moiety originating from the hemagglutinin protein, which can bind commercially available antibodies with high affinity. The anti-hemagglutinin peptide antibody, or more generally, a binding moiety that can specifically bind to the specific recognition moiety, can exist free in solution or can be attached to a solid support.
The term xe2x80x9csolid supportxe2x80x9d as used herein refers to a matrix composed of a material that does not dissolve in aqueous solutions. The solid support can be composed of such materials as carbohydrate and plastic materials. Many examples of commercially available solid supports are available to those skilled in the art. Examples of solid supports are latex and silica particles, plastics, agarose, cellulose, and polyethylene. Because solid supports with reactive chemical moieties present on their surfaces are commercially available or can be chemically synthesized using well known techniques in the art, components of the invention can be linked to the solid support either before or after the components are added to the sample comprising biological molecules under study. The components of the invention can be linked to the support either directly or by a spacer molecule. Examples of chemical linkages between solid supports and other molecules are well known to those skilled in the art (e.g., this information can be found in the Pierce catalogue). In addition, purified forms of biological molecules themselves can be linked to solid supports using techniques commonly known to those skilled in the art.
In another preferred embodiment the invention relates to the method of determining the ratio of biological molecules, where at least one of the components comprises a linkage to a solid support. In this preferred embodiment, the first component can comprise a linkage to a solid support, the second component can comprise a linkage to a solid support, or both the first and the second component can comprise linkages to solid supports. If both components comprise linkages to solid supports, the solid supports may be different types of solid supports, or are of the same type of solid support, but each type of component is linked to discrete solid support entities. The term xe2x80x9cdiscrete solid support entitiesxe2x80x9d as used herein refers to one component linked to one solid support and another type of component linked to another solid support, where the solid support composition may be the same or different.
In another preferred embodiment, the invention relates to the method of determining the ratio of biological molecules, where the second component has specific binding affinity for a complex comprising, consisting of, or consisting essentially of one biological molecule and the fist component. The invention is preferably practiced in the manner stated by this preferred embodiment when the concentration of the biological molecule exceeds the concentration of the second component to which that biological molecule specifically binds. The second component can bind a complex of two or more molecules when a binding region of the second component has specific binding affinity to a region on a biological molecule and an adjacent region on the first component. Examples of bifunctional organically synthesized molecules as well as antibodies that bind complexes exist in the art. See, e.g., U.S. application Ser. No. 08/071,203 filed Jun. 1, 1993, and U.S. application Ser. No. 08/458,901 filed Jun. 2, 1995.
In a preferred embodiment, the invention relates to the method of determining the ratio of biological molecules, where the first component comprises a binding moiety having specific binding affinity for each of the biological molecules of interest. Each of the molecules bind to the first component in a ratio related to their solution ratio. For example, a binding moiety may have specific binding affinity for a modified molecule and its related unmodified form: a binding moiety may have specific binding affinity for hemoglobin and its modified form, hemoglobin A1-C. The binding moiety may bind to the modified and unmodified forms of biological molecules with equal affinity or unequal affinity. If the two forms of the biological molecules bind to the first component with unequal affinity, a normalization factor can be determined to correct for the actual ratio of the biological molecules bound to the first component. Alternatively, the ratio can be simply determined using a standard curve constructed as described herein by example. It can be advantageous to select antibodies with unequal affinity to the biological molecules if it is preferred to preferentially bind one of the biological molecules. For example, a larger, dynamic range can be achieved when one biological molecule is one-half or less than the concentration of the other biological molecule.
In another preferred embodiment, the invention relates to the method of determining the ratio of biological molecules, where the first component comprises: (a) a first binding moiety having specific binding affinity for one biological molecule; and (b) a second binding moiety having specific binding affinity for another of the biological molecules of interest. In this preferred embodiment, the first component can bind each of the biological molecules of interest, but is constructed such that each of the biological molecules compete for it. Specifically, the first component may only bind to one of the biological molecules of interest at one time. The invention can also relate to a first component in which the first binding moiety has specific binding affinity for one biological molecule and a second binding moiety has specific binding affinity for one or more other biological molecules. In this manner, a ratio can be determined for one molecule to a family of molecules if desired. Preferably, the ratio is determined for one biological molecule to one other biological molecule.
In other preferred embodiments, the invention relates to the method of determining the ratio of biological molecules, where one or more of the binding moieties of the first component or second component are antibodies.
In yet another preferred embodiment the invention relates to the method of determining the ratio of biological molecules, where the biological molecules are occupied receptor and free receptor.
The term xe2x80x9cfree receptorxe2x80x9d as used herein refers to a molecule that functions by binding another molecule. A free receptor is a receptor molecule that is unbound by a ligand. A receptor molecule can exist on the surface of a cell or within the cell. Examples of receptors found on the surface of cells are mitogenic receptors (such as epidermal growth factor receptor and platelet derived growth factor receptor), metabolic receptors (such as insulin receptor and transferrin receptor), platelet aggregation receptors (such as glycoprotein IIbIIIa receptor), steroid receptors, and hormone receptors.
The term xe2x80x9coccupied receptorxe2x80x9d as used herein refers to a receptor that is bound by a ligand. The term xe2x80x9cligandxe2x80x9d refers to a molecule that binds to the receptor with high affinity. Examples of naturally occurring ligands of receptors are, for example, iron for the transferrin receptor, epidermal growth factor for the epidermal growth factor receptor, and fibrinogen or specific drugs, such as Reopro(copyright), for binding to the glycoprotein IIbIIIa receptor. The ligand may also be a synthetic ligand which binds with high affinity to the receptor. The term xe2x80x9chigh affinityxe2x80x9d as used herein in reference to a receptor-ligand interaction refers to a dissociation equilibrium binding constant between 1 xcexcM and 0.01 pM.
An example of a pharmaceutically relevant free/occupied receptor system relates to receptor glycoprotein IIbIIIa and its role in thrombosis. Thrombosis is the process in which red blood cells form a clot upon binding fibrinogen. Various drugs already in the market or entering the market can bind to the glycoprotein IIbIIIa receptor and block the clotting process. Methods set forth herein can determine the amount of the anti-clotting drug required to effectively block the clotting process.
In yet another preferred embodiment, the invention relates to the method of determining the ratio of biological molecules, where the biological molecules are hemoglobin and hemoglobin A1-C.
The term xe2x80x9chemoglobinxe2x80x9d as used herein refers to a protein molecule that transports oxygen in the blood of organisms. Hemoglobin exists at high concentrations in an organism""s blood.
The term xe2x80x9chemoglobin A1-Cxe2x80x9d refers to hemoglobin that is modified when the-glucose concentration is high in an organism""s blood stream. Hemoglobin is modified by glucose moieties when the concentration of glucose achieves a critical concentration in the bloodstream of an organism. Hemoglobin is glycosylated at higher levels in diabetic patients as compared to non-diabetic patients because diabetic patients"" blood contain abnormally high concentrations of glucose.
In another preferred embodiment, the invention relates to the method of determining the ratio of biological molecules, where the biological molecules are oxidized troponin I and reduced troponin I.
The term xe2x80x9creduced troponin Ixe2x80x9d as used herein refers to troponin I containing two cysteine moieties that are capable of undergoing intramolecular oxidation. The cysteine amino acids have side chains of formula xe2x80x94CH2xe2x80x94SH. Reduced troponin I can contain at least two cysteine residues. Components of the invention can be specific for the reduced form of troponin I since it exists in a different protein conformation than the oxidized form of troponin I.
The term xe2x80x9coxidized troponin Ixe2x80x9d as used herein refers to troponin I containing one or more cystine moieties in an oxidized form. Oxidized cystine amino acids have side chains of formula xe2x80x94CH2xe2x80x94Sxe2x88x92. Oxidized troponin I can contain at least one cystine residue that is in an oxidized form.
In yet another preferred embodiment, the invention relates to the method of determining the ratio of biological molecules where the first component is specific for both occupied receptor and free receptor, and where the second component is specific for: (a) occupied receptor; (b) free receptor; (c) a complex comprising, consisting essentially of, or consisting of occupied receptor and the first component; or (d) a complex comprising, consisting essentially of, or consisting of free receptor and the first component. As described herein, the free receptor may relate to glycoprotein IIbIIIa and the occupied receptor may relate to glycoprotein IIbIIIa bound to a drug.
The term xe2x80x9cspecific for both occupied receptor and free receptorxe2x80x9d as used herein refers to a component, preferably an antibody, of the invention that can bind to a receptor or to a component comprising the receptor whether or not it is free or occupied. This type of component does not discriminate against free or occupied receptor. This component, however, binds to a receptor with higher affinity than to other molecules.
The first component, which binds to both the free and occupied forms of receptor, is different than the second component, which specifically binds to one of the forms of the receptor in an unbound state or a bound state or one of the forms of the receptor in a complex with the first component. A second component that specifically binds to a complex comprising, consisting of, or consisting essentially of free receptor and the first component, for example, will not specifically bind to a complex comprising, consisting of, or consisting essentially of occupied receptor and the first component.
In another preferred embodiment, the invention relates to the method of determining the ratio of biological molecules, where the first component is specific for both hemoglobin and hemoglobin A1-C, and where the second component is specific for: (a) hemoglobin; (b) hemoglobin A1-C; (c) a complex comprising, consisting essentially of, or consisting of hemoglobin and the first component; or (d) a complex comprising, consisting essentially of, or consisting of hemoglobin A1-C and the first component.
The term xe2x80x9cspecific for both hemoglobin and hemoglobin A1-Cxe2x80x9d as used herein refers to a component, preferably an antibody, of the invention that combines to hemoglobin whether or not it is modified by glucose. or unmodified by glucose. This type of component does not discriminate against hemoglobin that is not modified by glucose and hemoglobin that is modified by glucose. This component, however, binds to hemoglobin with higher affinity than to other proteins.
A first component that specifically binds to both hemoglobin and hemoglobin A1-C is different than a second component that specifically binds a complex comprising, consisting of, or consisting essentially of hemoglobin A1-C and the first component. In addition, a second component that specifically binds a complex comprising, consisting of, or consisting essentially of hemoglobin and the first component, for example, will not specifically bind to a complex comprising, consisting of, or consisting essentially of hemoglobin A1-C and the first component.
In yet another preferred embodiment the invention relates to the method of determining the ratio of biological molecules, where the first component is specific for both oxidized troponin I and reduced troponin I and where the second component is specific for: (a) oxidized troponin I; (b) reduced troponin I; (c) a complex comprising, consisting essentially of, or consisting of oxidized troponin I and the first component; or (d) a complex comprising, consisting essentially of, or consisting of reduced troponin I and the first component.
The term xe2x80x9cspecific for both oxidized troponin I and reduced troponin Ixe2x80x9d as used herein refers to a component, preferably an antibody, of the invention that binds to troponin I whether or not it is oxidized or reduced. This type of component does not discriminate against oxidized or reduced troponin I. This component, however, binds to troponin I with higher affinity than to other proteins.
A first component that specifically binds to both oxidized and reduced troponin I is different than a second component that specifically binds a complex comprising, consisting of, or consisting essentially of oxidized troponin I and the first component. In addition, a second component that specifically binds a complex comprising, consisting of, or consisting essentially of oxidized troponin I and the first component, for example, will not specifically bind to a complex comprising, consisting of, or consisting essentially of reduced troponin I and the first component.
In another preferred embodiment the invention relates to a method of determining the ratio of biological molecules, further comprising the step of contacting the biological molecules with a third component. The third component is preferably added to a sample comprising the biological molecules after the first and second components have been added to the sample, but added before the free molecules are washed away or before the ratio of the biological molecules is determined. The third component has specific binding affinity for a complex comprising the first biological molecule and the second component.
The third component can bind to a complex comprising the first biological molecule and the second component when the third component binds adjacent regions located on the first biological molecule and the second component.
In yet another preferred embodiment the invention relates to a method of determining the ratio of biological molecules, where the third component comprises a specific recognition moiety. This specific recognition moiety can be utilized to bind the complex to a solid support. Examples of specific recognition moieties are disclosed herein. The specific recognition moiety linked to the third component can be the same moiety as the specific recognition moiety potentially linked to the second component, but is preferably a different moiety than the recognition moiety potentially linked to the second component.
In another preferred embodiment, the invention relates to the method of determining the ratio of biological molecules, further comprising the step of removing molecules that are not bound to the complex comprising a biological molecule, a first component, and a second component before determining the amount of this complex.
The term xe2x80x9cremovingxe2x80x9d as used herein refers to a method of separating molecules from those existing in a complex comprising, consisting of, or consisting essentially of a biological molecule, a first component, and a second component. This method can be accomplished by attaching the first or the second component to a solid support and washing away molecules that are not bound to either the first or second component. These techniques are well-known to those skilled in the art. See e.g., Harlo and Lane, Antibodies, a Laboratory Manual, 1989, Cold Spring Harbor Laboratories.
A person of ordinary skill in the art could readily adapt the concepts and components of the invention to a method that does not require a solid support. Homogeneous assay methods have been described in the art where the amount of a given biological molecule can be determined by the change in the fluorescence polarization of a component to which the biological molecule binds. Some homogeneous assay techniques applicable to this invention are described in WO94/24559, U.S. Pat. Nos. 3,817,837 and 3,935,074, and in Clin. Chem. 32, 1637-1641, (1986) incorporated herein by reference in their entirety including any references and diagrams. Thus, changes in the physical parameters of the components of the invention (e.g., fluorescence polarization or absorbance or wavelength) could be monitored when biological molecules bind to them. These changes in physical parameters can be used to directly determine the ratio of biological molecules without the use of a solid support.
In another preferred embodiment, the invention relates to the method of determining the ratio of biological molecules further comprising the step of comparing the amount of the complex comprising a biological molecule, a first component, and a second component to a standard curve, where the standard curve relates the amount of this complex to the ratio of the biological molecules. This complex could further comprise a third component, where the third component has specific binding affinity for a complex comprising the first biological molecule and the second component. In addition, the third component can comprise a specific recognition moiety.
The term xe2x80x9cstandard curvexe2x80x9d as used herein refers to a measured relationship between the ratio of biological molecules to the amount of the complex comprising a biological molecule, a first component, and a second component. This complex could further comprise a third component, where the third component has specific binding affinity for a complex comprising the first biological molecule and the second component. In addition, the third component can comprise a specific recognition moiety. The amount of the complex can be quantified by a signal generated by a reporter molecule linked to one of the components of the invention. The relationship between the ratio and the signal in a standard curve, for example, may be linear or obey a non-linear function. The standard curve can be generated by measuring the signal generated by the methods of the invention for samples containing known ratios of biological molecules. These methods are described by example herein.
The term xe2x80x9csignalxe2x80x9d as used herein refers to a spectroscopic or chemical change caused by a reporter molecule attached to either a component of the invention or another component used to detect a complex comprising a biological molecule, a first component, and a second component. As described herein, the signal can, for example, take the form of a fluorescence emission, a change in the wavelength of a fluorescence emission, an absorbance measurement, a change in infrared wavelength, or a change in the pH of the solution.
The term xe2x80x9ccomparingxe2x80x9d as used herein, in reference to a standard curve, refers to extrapolating the ratio of biological molecules from a standard curve by using the amount of the second component bound to the biological molecules of interest. Because the standard curve relates the ratio of biological molecules to the signal generated by the method of the invention, applying a signal measurement to the standard curve can generate an estimated ratio of the biological molecules.
In another preferred embodiment, the invention relates to the method of determining the ratio of biological molecules, further comprising the step of comparing the signal generated from a reporter molecule to a standard curve. The standard curve can relate the amount of the reporter molecule to the ratio of the biological molecules.
In another aspect, the invention relates to a method for determining one or more solution ratios of three or more biological molecules. This method for determining the solution ratios of one or more biological molecules comprises the steps of: (a) contacting the biological molecules with (i) a first component having specific binding affinity for each of the biological molecules, where the biological molecules bind to the first component in a binding ratio related to the solution ratio of the biological molecules; (ii) a second component having specific binding affinity for a first biological molecule of the biological molecules; (iii) a different second component having specific binding affinity for a second biological molecule of the biological molecules; and (b) determining the amount of a complex comprising the first biological molecule, the first component, and the second component or the amount of a complex comprising the second biological molecule, the first component, and the different second component as a measure of the solution ratio.
In a preferred embodiment the invention relates to the method of determining the ratios of three or more biological molecules, wherein at least one of the components comprises an antibody.
In another preferred embodiment the invention relates to the method of determining the ratios of three or more biological molecules, wherein at least one of the components comprises a specific recognition moiety.
In yet another preferred embodiment the invention relates to the method of determining the ratios of three or more biological molecules, wherein at least one of the components comprises a linkage to a solid support.
In another preferred embodiment the invention relates to the method of determining the ratios of three or more biological molecules, wherein at least one of the components comprises a reporter molecule.
In yet another preferred embodiment the invention relates to the method of determining the ratios of three or more biological molecules, where the biological molecules are activated platelets, free glycoprotein IIbIIIa receptor, occupied glycoprotein IIbIIIa receptor, and P-selectin.
The term xe2x80x9cactivated plateletsxe2x80x9d as used herein refers to biological process of forming a thrombis. Inactive platelets and activated platelets express the protein glycophorin on the cell surface. Inactive platelets also express free glycoprotein IIbIIIa receptor. This receptor can bind fibrinogen, which activates the platelets and induces them to form a thrombis. Activated platelets, but not inactive platelets, express the protein P-selectin on the cell surface. Molecules that bind and occupy the glycoprotein IIbIIIa receptor can block the binding of fibrinogen to the receptor and thereby inhibit the activation of platelets and inhibit the formation of thrombis clots. The methods provided herein by example an determine the ratio of occupied to free glycoprotein IIbIIIa receptor and the ratio of activated to inactive platelets.
The term xe2x80x9cinactive plateletsxe2x80x9d as used herein refers to platelets that have the potential to be activated but have not yet been activated because the proper activation signal has not activated them or because a drug is bound to the glycoprotein IIbIIIa receptor and blocking the activation signal.
The term xe2x80x9cfree glycoprotein IIbIlIa receptorxe2x80x9d as used herein refers to the receptor that is not bound by fibrinogen or by any drug molecules.
The term xe2x80x9coccupied glycoprotein IIbIlIa receptorxe2x80x9d as used herein refers to the receptor that is bound by fibrinogen or by drug molecules which inhibit the activation of platelets.
The term xe2x80x9cglycophorinxe2x80x9d as used herein refers to a protein that is expressed on the surface of both inactive and activated platelets.
The term xe2x80x9cP-selectinxe2x80x9d as used herein refers to a protein that is expressed on the surface of activated platelets.
In other preferred embodiments the invention relates to the method for determining the ratio of three or more biological molecules, where the amount of the complex comprising the first biological molecule, the first component, and the second component is a measure of the solution ratio of free glycoprotein IIbIIIa receptor to occupied glycoprotein IIbIIIa receptor. In addition, the amount of the complex comprising the second biological molecule, the first component, and the different second component is a measure of the solution ratio of activated platelets to inactive platelets.
In a preferred embodiment the invention relates to the method of determining the ratios of three or more biological molecules, where the second component has specific binding affinity for either free glycoprotein IIbIIIa receptor or occupied glycoprotein IIbIIIa receptor, and the different second component has specific binding affinity for P-selectin.
In another preferred embodiment the invention relates to the method of determining the ratios of three or more biological molecules, where the method further comprises another different second component having specific binding affinity for a third biological molecule. This other different second component can comprise a specific recognition moiety.
In another preferred embodiment the invention relates to the method of determining the ratios of three or more biological molecules, where the first component is specific for glycophorin, where the second component has specific binding affinity for free glycoprotein IIbIIIa receptor, where the different second component has specific binding affinity for P-selectin, and where the other different second component has specific binding affinity for occupied glycoprotein IIbIIIa receptor.
In an aspect that bears on the foregoing embodiments and aspects of the invention, components of the invention can have specific binding affinity to two or more biological molecules, where the biological molecules are (a) related, (b) not related, or (c) related and not related.
Examples of components that have specific binding affinity for biological molecules that are not related are illustrated in FIG. 2. FIG. 2A illustrates a component that comprises a single antibody, where the antibody has specific binding affinity for molecule A and molecule B. Molecule A and molecule B may not be related, and binding of molecule A prevents binding of molecule B. Similarly, binding of molecule B prevents the binding of molecule A to the component.
FIG. 2B illustrate another component that can bind biological molecules that are not related, where the component comprises two antibodies, each having specific binding affinity for one biological molecule, either molecule A or molecule B. The antibodies of the component can be arranged in space such that binding of molecule A to the component prevents the binding of molecule B to the component, and binding of molecule B prevents binding of molecule A.
The term xe2x80x9crelated biological moleculesxe2x80x9d as used herein can refer to biological molecules having significant structural similarity to one another. Such related molecules can have substantial amino acid sequence identity between one another or can have substantial nucleic acid sequence identity with one another. Amino acid sequence identity and nucleic acid sequence identity are well known in the art. Examples of related biological molecules are isoforms of a given biological protein, such as hemoglobin and hemoglobin A1-C, oxidized and reduced troponin I, occupied and unoccupied cell surface receptors, or occupied and unoccupied cell receptors.
Biological molecules that are not related may have structural dissimilarities. Such structural dissimilarities may be reflected in amino acid sequence identities and nucleic acid sequence identities that are lower than those for related biological molecules. Examples of biological molecules that are not related are hemoglobin and troponin I, or myoglobin and troponin I. These examples are not meant to be limiting and the invention relates to any biological molecules that are not related.
In FIGS. 1A, 1B, 1C, and 1D, molecules A, B, C, and D may be related, not related, or a mixture thereof. In applications of the invention that concern the determination of one or more ratios of non-related molecules, components illustrated in FIG. 2 can be utilized to bind any non-related biological molecules. Such components illustrated in FIG. 2 can be utilized as components for binding non-related biological molecules in any one of the schemes illustrated in FIGS. 1A, 1B, 1C, and 1D. For example, in methods for determining the ratio of non-related biological molecules, component 1 of FIG. 1A can resemble the component illustrated in FIG. 2, where the component can bind any of the non-related biological molecules, and where binding of one molecule precludes the binding of another non-related molecule.
In another aspect, the invention relates to a kit for determining the ratio of biological molecules. The kit comprises the following elements: (a) a first component having specific binding affinity to the biological molecules, where the biological molecules bind to the first component in an amount that is proportional to their ratio in the sample for the first component; and (b) a second component having specific binding affinity for one or more of the biological molecules. The kit may also comprise a label or Food and Drug Administration approved protocol indicating the steps for determining the ratio.
The term xe2x80x9ckitxe2x80x9d as used herein refers to a packaged product comprising components of the invention used to determine the ratio of biological molecules. The kit preferably comprises a box or container that holds the components of the kit. The box or container is affixed with a label or a Food and Drug Administration approved protocol. The box or container holds components to the invention which are preferably contained within plastic, polyethylene, polypropylene, ethylene, or propylene vessels. The vessels can be capped tubes or bottles.
The term xe2x80x9clabelxe2x80x9d as used herein can refer to an indicator on the outside of a kit. The label can be constructed from material or another material such as plastic.
Alternatively, the term xe2x80x9clabelxe2x80x9d as used herein can be used to describe a xe2x80x9csignal generatorxe2x80x9d or xe2x80x9csignal generating elementxe2x80x9d or xe2x80x9creporter molecule.xe2x80x9d
In a preferred embodiment, the invention relates to the kit for determining the ratio of biological molecules, where at least one of the components comprises an antibody. In this preferred embodiment, the first component can comprise an antibody, the second component can comprise an antibody, or the first component and the second component can comprise antibodies. Thus, the method can utilize a first component that comprises an antibody and a second component that comprises another type of polypeptide or organic molecule. Alternatively, the method may relate to two components that comprise antibodies.
In yet another preferred embodiment, the invention relates to the kit for determining the ratio of biological molecules, where at least one of the components comprises a reporter molecule. In this preferred embodiment, the first component can comprise a reporter molecule, the second component can comprise a reporter molecule, or the first component and the second component can comprise reporter molecules. If both components comprise reporter molecules, the reporter molecules may be the same types of molecules, or preferably, different types of reporter molecules.
In another preferred embodiment the invention relates to the kit for determining the ratio of biological molecules, where at least one component comprises a specific recognition moiety. In this preferred embodiment, the first component can comprise a specific recognition moiety, the second component can comprise a specific recognition moiety, or the first component and the second component can comprise specific recognition moieties. If both components comprise specific recognition moieties, the specific recognition moieties may be different from one another or the same recognition moiety.
In another preferred embodiment the invention relates to the kit for determining the ratio of biological molecules, where at least one of the components comprises a linkage to a solid support. In this preferred embodiment, the first component can comprise a linkage to a solid support, the second component can comprise a linkage to a solid support, or both the first and the second component can comprise linkages to solid supports. If both components comprise linkages to solid supports, the solid supports may be different types of solid supports, or are of the same type of solid support, but each type of component is linked to discrete solid support entities. The term xe2x80x9cdiscrete solid support entitiesxe2x80x9d as used herein refers to one component linked to one solid support and another type of component linked to another solid support, where the solid support composition may be the same of different.
In another preferred embodiment, the invention relates to the kit for determining the ratio of biological molecules, where the second component has specific binding affinity for a complex comprising, consisting of, or consisting essentially of one biological molecule and the fist component. The invention is preferably practiced in the manner stated by this preferred embodiment when the concentration of an unbound biological molecule exceeds the concentration of the second component to which that biological molecule specifically binds. The second component can bind a complex of one or more molecules when a binding region of the second component has specific binding affinity to a region on a biological molecule and an adjacent region on the first component. Examples of bifunctional organically synthesized molecules as well as antibodies that bind complexes exist in the art.
In a preferred embodiment, the invention relates to the kit for determining the ratio of biological molecules, where the first component comprises a binding moiety having specific binding affinity for each of the biological molecules of interest. Each of the molecules bind to the first component in a ratio related to their solution ratio. For example, a binding moiety may have specific binding affinity for a modified molecule and its related unmodified form: a binding moiety may have specific binding affinity for hemoglobin and its modified form, hemoglobin A1-C. The binding moiety may bind to the modified and unmodified forms of biological molecules with equal affinity or unequal affinity. If the two forms of the biological molecules bind to the first component with unequal affinity, a normalization factor can be determined to correct for the actual ratio of the biological molecules bound to the first component. Alternatively, the ratio can be simply determined using a standard curve constructed as described herein by example.
In another preferred embodiment, the invention relates to the kit for determining the ratio of biological molecules, where the first component comprises: (a) a first binding moiety having specific binding affinity for one biological molecule; and (b) a second binding moiety having specific binding affinity for another of the biological molecules of interest. In this preferred embodiment, the first component can bind each of the biological molecules of interest, but is constructed such that each of the biological molecules compete for it. Specifically, the biological molecules may only bind to one of the biological molecules of interest at one time. The invention can also relate to a first component in which the first binding moiety has specific binding affinity for one biological molecule and a second binding moiety has specific binding affinity for one or more other biological molecules. In this manner, a ratio can be determined for one molecule to a family of molecules if desired. Preferably, the ratio is determined for one biological molecule to one other biological molecule.
In other preferred embodiments, the invention relates to the kit for determining the ratio of biological molecules, where one or more of the binding moieties of the first component or second component are antibodies.
In yet another preferred embodiment, the invention relates to the kit for determining the ratio of biological molecules where the first component is specific for both occupied receptor and free receptor, and where the second component is specific for: (a) occupied receptor; (b) free receptor; (c) a complex comprising, consisting essentially of, or consisting of occupied receptor and the first component; or (d) a complex comprising, consisting essentially of, or consisting of free receptor and the first component.
In another preferred embodiment, the invention relates to the kit for determining the ratio of biological molecules, where the first component is specific for both hemoglobin and hemoglobin A1-C, and where the second component is specific for: (a) hemoglobin; (b) hemoglobin A1-C; (c) a complex comprising, consisting essentially of, or consisting of hemoglobin and the first component; or (d) a complex comprising, consisting essentially of, or consisting of hemoglobin A1-C and the first component.
In yet another preferred embodiment the invention relates to the kit for determining the ratio of biological molecules, where the first component is specific for both oxidized troponin I and reduced troponin I and where the second component is specific for: (a) oxidized troponin I; (b) reduced troponin I; (c) a complex comprising, consisting essentially of, or consisting of oxidized troponin I and the first component; or (d) a complex comprising, consisting essentially of, or consisting of reduced troponin I and the first component.
Another preferred embodiment relates to the kit for determining the ratio of biological molecules, further comprising a third component, where the third component has specific binding affinity for a complex comprising the first biological molecule and the second component. The third component may comprise a specific recognition moiety.
In another preferred embodiment the invention relates to the kit for determining the ratio of biological molecules, where the kit further comprises a different second component, and where the different second component has specific binding affinity for a second biological molecule. The different second component can comprise a specific recognition moiety.
In yet another preferred embodiment the invention relates to the kit for determining the ratio of biological molecules, where the kit further comprises another different second component, and where this other different second component has specific binding affinity for a third biological molecule. This other different second component can comprise a specific recognition moiety.
In another preferred embodiment the invention relates to the kit for determining the ratio of biological molecules, where the first component is specific for glycophorin, where the second component has specific binding affinity for either free glycoprotein IlbIIIa receptor or occupied glycoprotein IIbIIIa receptor, and where the third component has specific binding affinity for P-selectin.
Another preferred embodiment relates to the kit for determining the ratio of biological molecules further comprising the biological molecules themselves. The biological molecules are in a purified form suitable for determining the ratio of the biological molecules.
The term xe2x80x9cpurified formxe2x80x9d as used herein refers to the degree of heterogeneity of the biological molecules. Multiple purification processes are known to those skilled in the art. An example of a purification process is high performance liquid chromatography using ion exchange, size exclusion, and hydrophobic techniques. These processes can be applied to proteinaceous molecules as well as organic molecules.
The term xe2x80x9csuitable for determining the ratioxe2x80x9d as used herein refers to a purified form of the biological molecules that yields reproducible results in the method described on the label of the kit. The term refers to a level of purity such that other molecules do not significantly interfere with the binding between the components of the invention and the biological molecules of the invention.
In yet another preferred embodiment, the invention relates to a kit for determining the ratio of biological molecules where the biological molecules are occupied receptor and free receptor.
In another preferred embodiment, the invention relates to a kit for determining the ratio of biological molecules where the biological molecules are hemoglobin and hemoglobin A1-C.
In a preferred embodiment the invention relates to a kit for determining the ratio of biological molecules where the biological molecules are oxidized troponin I and reduced troponin I.
In another preferred embodiment the invention relates to the kit for determining the ratio of biological molecules where the biological molecules are glycophorin, free glycoprotein IIbIIIa receptor, occupied glycoprotein IIbIIa receptor, and P-selectin.
The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims.
The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention and from the claims.